E and M SARS-CoV-2 membrane protein expression and enrichment with plant lipid droplets.

Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs). LDs offer an ideal environment for folding hydrophobic proteins and aid in their purification through flotation. We tested various protein fusions with different linkers and tags and used three dimensional structure predictions to assess their effects. E and M mostly localized in the ER in N. benthamiana leaves but E could be targeted to LDs in oil accumulating tobacco when fused with oleosin, a LD integral protein. In Camelina sativa seeds, E and M were however found associated with purified LDs. By enhancing the accumulation of E and M within LDs through oleosin, we enriched these proteins in the purified floating fraction. This strategy provides an alternative approach for efficiently producing and purifying hydrophobic pharmaceuticals and vaccines using plant systems.

Fine-mapping and evolutionary history of R-BPMV, a dominant resistance gene to Bean pod mottle virus in Phaseolus vulgaris L.

KEY MESSAGE: R-BPMV is located within a recently expanded TNL cluster in the Phaseolus genus with suppressed recombination and known for resistance to multiple pathogens including potyviruses controlled by the I gene. Bean pod mottle virus (BPMV) is a comovirus that infects common bean and legumes in general. BPMV is distributed throughout the world and is a major threat on soybean, a closely related species of common bean. In common bean, BAT93 was reported to carry the R-BPMV resistance gene conferring resistance to BPMV and linked with the I resistance gene. To fine map R-BPMV, 182 recombinant inbred lines (RILs) derived from the cross BAT93 × JaloEEP558 were genotyped with polymerase chain reaction (PCR)-based markers developed using genome assemblies from G19833 and BAT93, as well as BAT93 BAC clone sequences. Analysis of RILs carrying key recombination events positioned R-BPMV to a target region containing at least 16 TIR-NB-LRR (TNL) sequences in BAT93. Because the I cluster presents a suppression of recombination and a large number of repeated sequences, none of the 16 TNLs could be excluded as R-BPMV candidate gene. The evolutionary history of the TNLs for the I cluster were reconstructed using microsynteny and phylogenetic analyses within the legume family. A single I TNL was present in Medicago truncatula and lost in soybean, mirroring the absence of complete BPMV resistance in soybean. Amplification of TNLs in the I cluster predates the divergence of the Phaseolus species, in agreement with the emergence of R-BPMV before the separation of the common bean wild centers of diversity. This analysis provides PCR-based markers useful in marker-assisted selection (MAS) and laid the foundation for cloning of R-BPMV resistance gene in order to transfer the resistance into soybean.

R-BPMV-Mediated Resistance to Bean pod mottle virus in Phaseolus vulgaris L. Is Heat-Stable but Elevated Temperatures Boost Viral Infection in Susceptible Genotypes.

In the context of climate change, elevated temperature is a major concern due to the impact on plant-pathogen interactions. Although atmospheric temperature is predicted to increase in the next century, heat waves during summer seasons have already become a current problem. Elevated temperatures strongly influence plant-virus interactions, the most drastic effect being a breakdown of plant viral resistance conferred by some major resistance genes. In this work, we focused on the R-BPMV gene, a major resistance gene against Bean pod mottle virus in Phaseolus vulgaris. We inoculated different BPMV constructs in order to study the behavior of the R-BPMV-mediated resistance at normal (20 °C) and elevated temperatures (constant 25, 30, and 35 °C). Our results show that R-BPMV mediates a temperature-dependent phenotype of resistance from hypersensitive reaction at 20 °C to chlorotic lesions at 35 °C in the resistant genotype BAT93. BPMV is detected in inoculated leaves but not in systemic ones, suggesting that the resistance remains heat-stable up to 35 °C. R-BPMV segregates as an incompletely dominant gene in an F2 population. We also investigated the impact of elevated temperature on BPMV infection in susceptible genotypes, and our results reveal that elevated temperatures boost BPMV infection both locally and systemically in susceptible genotypes.

A common bean truncated CRINKLY4 kinase controls gene-for-gene resistance to the fungus Colletotrichum lindemuthianum.

Identifying the molecular basis of resistance to pathogens is critical to promote a chemical-free cropping system. In plants, nucleotide-binding leucine-rich repeat constitute the largest family of disease resistance (R) genes, but this resistance can be rapidly overcome by the pathogen, prompting research into alternative sources of resistance. Anthracnose, caused by the fungus Colletotrichum lindemuthianum, is one of the most important diseases of common bean. This study aimed to identify the molecular basis of Co-x, an anthracnose R gene conferring total resistance to the extremely virulent C. lindemuthianum strain 100. To that end, we sequenced the Co-x 58 kb target region in the resistant JaloEEP558 (Co-x) common bean and identified KTR2/3, an additional gene encoding a truncated and chimeric CRINKLY4 kinase, located within a CRINKLY4 kinase cluster. The presence of KTR2/3 is strictly correlated with resistance to strain 100 in a diversity panel of common beans. Furthermore, KTR2/3 expression is up-regulated 24 hours post-inoculation and its transient expression in a susceptible genotype increases resistance to strain 100. Our results provide evidence that Co-x encodes a truncated and chimeric CRINKLY4 kinase probably resulting from an unequal recombination event that occurred recently in the Andean domesticated gene pool. This atypical R gene may act as a decoy involved in indirect recognition of a fungal effector.

Genetic resistance against viruses in Phaseolus vulgaris L.: State of the art and future prospects.

Viruses are obligate parasites that replicate intracellularly in many living organisms, including plants. Consequently, no chemicals are available that target only the virus without impacting host cells or vector organisms. The use of natural resistant varieties appears as the most reliable control strategy and remains the best and cheapest option in managing virus diseases, especially in the current ecological context of preserving biodiversity and environment in which the use of phytosanitary products becomes limited. Common bean is a grain legume cultivated mainly in Africa and Central-South America. Virus diseases of common bean have been extensively studied both by breeders to identify natural resistance genes in existing germplasms and by pathologists to understand the molecular bases of plant-virus interactions. Here we present a critical review in which we synthesize previous and recent information concerning 1) main viruses causing diseases in common bean, 2) genetic resistance to viruses in common bean, 3) the different resistance phenotypes observed and more particularly the effect of temperature, 4) the molecular bases of resistance genes to viruses in common bean, and 5) future prospects using transgenic-engineered resistant lines.

Virus-Induced Gene Silencing (VIGS) and Foreign Gene Expression in Pisum sativum L. Using the "One-Step" Bean pod mottle virus (BPMV) Viral Vector.

Plant viral vectors have been developed to facilitate gene function studies especially in plant species not amenable to traditional mutational or transgenic modifications. In the Fabaceae plant family, the most widely used viral vector is derived from Bean pod mottle virus (BPMV). Originally developed for overexpression of foreign proteins and VIGS studies in soybean, we adapted the BPMV-derived vector for use in other legume species such as Phaseolus vulgaris and Pisum sativum. Here, we describe a protocol for efficient protein expression and virus-induced gene silencing (VIGS) in Pisum sativum leaves and roots using the "one-step" Bean pod mottle virus (BPMV) viral vector.

Bean pod mottle virus: a new powerful tool for functional genomics studies in Pisum sativum.

Pea (Pisum sativum L.) is an important legume worldwide. The importance of pea in arable rotations and nutritional value for both human and animal consumption have fostered sustained production and different studies to improve agronomic traits of interest. Moreover, complete sequencing of the pea genome is currently underway and will lead to the identification of a large number of genes potentially associated with important agronomic traits. Because stable genetic transformation is laborious for pea, virus-induced gene silencing (VIGS) appears as a powerful alternative technology for determining the function of unknown genes. In this work, we present a rapid and efficient viral inoculation method using DNA infectious plasmids of Bean pod mottle virus (BPMV)-derived VIGS vector. Six pea genotypes with important genes controlling biotic and/or abiotic stresses were found susceptible to BPMV carrying a GFP reporter gene and showed fluorescence in both shoots and roots. In a second step, we investigated 37 additional pea genotypes and found that 30 were susceptible to BPMV and only 7 were resistant. The capacity of BPMV to induce silencing of endogenes was investigated in the most susceptible genotype using two visual reporter genes: PsPDS and PsKORRIGAN1 (PsKOR1) encoding PHYTOENE DESATURASE and a 1,4-ß-D-glucanase, respectively. The features of the 'one-step' BPMV-derived VIGS vector include (i) the ease of rub-inoculation, without any need for biolistic or agro-inoculation procedures, (ii) simple cost-effective procedure and (iii) noninterference of viral symptoms with silencing. These features make BPMV the most adapted VIGS vector in pea to make low- to high-throughput VIGS studies.

Development of molecular markers linked to disease resistance genes in common bean based on whole genome sequence.

Common bean (Phaseolus vulgaris) is the most important grain legume for direct human consumption in the world, particularly in developing countries where it constitutes the main source of protein. Unfortunately, common bean yield stability is constrained by a number of pests and diseases. As use of resistant genotypes is the most economic and ecologically safe means for controlling plant diseases, efforts have been made to genetically characterize resistance genes (R genes) in common bean. Despite its agronomic importance, genomic resources available in common bean were limited until the recent sequencing of common bean genome (Andean genotype G19833). Besides allowing the annotation of Nucleotide Binding-Leucine Rich Repeat (NB-LRR) encoding gene family, which is the prevalent class of disease R genes in plants, access to the whole genome sequence of common bean can be of great help for intense selection to increase the overall efficiency of crop improvement programs using marker-assisted selection (MAS). This review presents the state of the art of common bean NB-LRR gene clusters, their peculiar location in subtelomeres and correlation with genetically characterized monogenic R genes, as well as how the availability of the whole genome sequence can boost the development of molecular markers for MAS.

The "one-step" Bean pod mottle virus (BPMV)-derived vector is a functional genomics tool for efficient overexpression of heterologous protein, virus-induced gene silencing and genetic mapping of BPMV R-gene in common bean (Phaseolus vulgaris L.).

BACKGROUND: Over the last two years, considerable advances have been made in common bean (Phaseolus vulgaris L.) genomics, especially with the completion of the genome sequence and the availability of RNAseq data. However, as common bean is recalcitrant to stable genetic transformation, much work remains to be done for the development of functional genomics tools adapted to large-scale studies. RESULTS: Here we report the successful implementation of an efficient viral vector system for foreign gene expression, virus-induced gene silencing (VIGS) and genetic mapping of a BPMV resistance gene in common bean, using a "one-step" BPMV vector originally developed in soybean. With the goal of developing this vector for high-throughput VIGS studies in common bean, we optimized the conditions for rub-inoculation of infectious BPMV-derived plasmids in common bean cv. Black Valentine. We then tested the susceptibility to BPMV of six cultivars, and found that only Black Valentine and JaloEEP558 were susceptible to BPMV. We used a BPMV-GFP construct to detect the spatial and temporal infection patterns of BPMV in vegetative and reproductive tissues. VIGS of the PHYTOENE DESATURASE (PvPDS) marker gene was successfully achieved with recombinant BPMV vectors carrying fragments ranging from 132 to 391 bp. Finally, we mapped a gene for resistance to BPMV (R-BPMV) at one end of linkage group 2, in the vicinity of a locus (I locus) previously shown to be involved in virus resistance. CONCLUSIONS: The "one-step" BPMV vector system therefore enables rapid and simple functional studies in common bean, and could be suitable for large-scale analyses. In the post-genomic era, these advances are timely for the common bean research community.

VIGS technology: an attractive tool for functional genomics studies in legumes.

Legume species are among the most important crops worldwide. In recent years, six legume genomes have been completely sequenced, and there is now an urgent need for reverse-genetics tools to validate genes affecting yield and product quality. As most legumes are recalcitrant to stable genetic transformation, virus-induced gene silencing (VIGS) appears to be a powerful alternative technology for determining the function of unknown genes. VIGS technology is based on the property of plant viruses to trigger a defence mechanism related to post-transcriptional gene silencing (PTGS). Infection by a recombinant virus carrying a fragment of a plant target gene will induce homology-dependent silencing of the endogenous target gene. Several VIGS systems have been developed for legume species since 2004, including those based on Bean pod mottle virus, Pea early browning virus, and Apple latent spherical virus, and used in reverse-genetics studies of a wide variety of plant biological processes. In this work, we give an overview of the VIGS systems available for legumes, and present their successful applications in functional genomics studies. We also discuss the limitations of these VIGS systems and the future challenges to be faced in order to use VIGS to its full potential in legume species.